Hydrogenases display a wide range of catalytic rates and biases in reversible hydrogen gas oxidation catalysis. The interactions of the iron–sulfur-containing catalytic site with the local protein ...environment are thought to contribute to differences in catalytic reactivity, but this has not been demonstrated. The microbe Clostridium pasteurianum produces three FeFe-hydrogenases that differ in “catalytic bias” by exerting a disproportionate rate acceleration in one direction or the other that spans a remarkable 6 orders of magnitude. The combination of high-resolution structural work, biochemical analyses, and computational modeling indicates that protein secondary interactions directly influence the relative stabilization/destabilization of different oxidation states of the active site metal cluster. This selective stabilization or destabilization of oxidation states can preferentially promote hydrogen oxidation or proton reduction and represents a simple yet elegant model by which a protein catalytic site can confer catalytic bias.
Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we ...report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.
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► Super-resolution microscopy is extended to densely labeled and multicellular specimens ► Thin, planar excitation enables multicolor imaging of 3D dynamics over extended times ► High axial resolution reveals complex 3D dynamics inaccessible to confocal microscopy ► In vivo karyotyping reveals specific chromosome arrangements during cell division
By combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy, 3D dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points.
Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used ...high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.
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•4D imaging elucidates the order of events leading to secretion•Actin depletion initiates events leading to centrosome polarization and secretion•Lattice light-sheet imaging reveals a rearward flow of actin away from the synapse•Both centrosome and granules are delivered to an area of membrane depleted of actin
The key sequence of events by which cytotoxic T lymphocytes establish an immunological synapse to kill target cells remains unclear. Griffiths and colleagues have resolved this by using high-resolution 4D imaging, revealing a critical role for actin reorganization in initiating centrosome polarization and granule secretion.
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ ...visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
Remarkably, forces within a neuron can extend its axon to a target that could be meters away. The two main cytoskeleton components in neurons are microtubules, which are mostly bundled along the axon ...shaft, and actin filaments, which are highly enriched in a structure at the axon distal tip, the growth cone. Neurite extension has been thought to be driven by a combination of two forces: pushing via microtubule assembly, and/or pulling by an actin-driven mechanism in the growth cone 1, 2. Here we show that a novel mechanism, sliding of microtubules against each other by the microtubule motor kinesin-1, provides the mechanical forces necessary for initial neurite extension in Drosophila neurons. Neither actin filaments in the growth cone nor tubulin polymerization is required for initial outgrowth. Microtubule sliding in neurons is developmentally regulated and is suppressed during neuronal maturation. As kinesin-1 is highly evolutionarily conserved from Drosophila to humans, it is likely that kinesin-1-powered microtubule sliding plays an important role in neurite extension in many types of neurons across species.
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•Microtubules actively slide in young, growing neurons•Microtubule sliding drives neurite membrane protrusion•Microtubule sliding is powered by kinesin-1•Microtubule sliding is developmentally regulated
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most ...commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.
We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest ...monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
This critical review provides an overview of the continually expanding family of fluorescent proteins (FPs) that have become essential tools for studies of cell biology and physiology. Here, we ...describe the characteristics of the genetically encoded fluorescent markers that now span the visible spectrum from deep blue to deep red. We identify some of the novel FPs that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells, and describe how many different optical methods can be combined with the FPs to provide quantitative measurements in living systems (227 references).
Adding to the super-resolution arsenal
Structured illumination microscopy (SIM) uses light intensities that are orders of magnitude lower than other super-resolution methods. SIM is also far faster ...over cellular-sized fields of view. Li
et al.
used two approaches to improve the resolution of SIM to allow live cell imaging of dynamic cellular processes, including endocytosis and cytoskeleton remodeling. The contrast in performance between SIM and other techniques is due to a few key differences. Defining the practical resolution at the limited signal-to-noise ratios necessary for live cell imaging will require better imaging metrics.
Science
, this issue
10.1126/science.aab3500
Super-resolution imaging of fast dynamic processes in living cells is facilitated by improvements to structured illumination microscopy.
INTRODUCTION
Various methods of super-resolution (SR) fluorescence microscopy have the potential to follow the dynamic nanoscale interactions of specific macromolecular assemblies in living cells. However, this potential is often left unfulfilled, either owing to the method’s inability to follow these processes at the speeds dictated by nature or because they require intense light that can substantially perturb the very physiology one hopes to study. An exception is structured illumination microscopy (SIM), which can image live cells far faster and with orders of magnitude less light than required for other SR approaches. However, SIM’s resolution is usually limited to only a twofold gain beyond conventional optical microscopes, or ~100 nm with visible light.
RATIONALE
We endeavored to find ways to extend SIM to the sub-100-nm regime while retaining, to the greatest extent possible, the advantages that make it the preferred SR method for live-cell imaging. Our first solution used an ultrahigh numerical aperture (NA) lens and total internal reflection fluorescence (TIRF) to achieve 84-nm resolution at subsecond acquisition speeds over hundreds of time points in multiple colors near the basal plasma membrane. Our second exploited the spatially patterned activation of a recently developed, reversibly photoswitchable fluorescent protein to reach 45- to 62-nm resolution, also at subsecond acquisition, over ∼10 to 40 time points.
RESULTS
We used high-NA TIRF-SIM to image the dynamic associations of cortical filamentous actin with myosin IIA, paxillin, or clathrin, as well as paxillin with vinculin and clathrin with transferrin receptors. Thanks to the combination of high spatial and temporal resolution, we were able to measure the sizes of individual clathrin-coated pits through their initiation, growth, and internalization. We were also able to relate pit size to lifetime, identify and characterize localized hot spots of pit generation, and describe the interaction of actin with clathrin and its role in accelerating endocytosis. With nonlinear SIM by use of patterned activation (PA NL-SIM), we monitored the remodeling of the actin cytoskeleton and the dynamics of caveolae at the cell surface. By combining TIRF-SIM and PA NL-SIM for two-color imaging, we followed the dynamic association of actin with α-actinin in expanding filopodia and membrane ruffles and characterized shape changes in and the transport of early endosomes. Last, by combining PA NL-SIM with lattice light sheet microscopy, we observed, in three dimensions and across the entire volume of whole cells, the dynamics of the actin cytoskeleton, the fusion and fission of mitochondria, and the trafficking of vesicles to and from the Golgi apparatus, each at axial resolution fivefold better than that of conventional widefield microscopy.
In addition, through direct experimental comparisons, we demonstrated that the resolution for our methods is comparable with or better than other SR approaches yet allowed us to image at far higher speeds, and for far longer durations. To understand why this is so, we developed a detailed theoretical model showing that our methods transmit the information encoded in spatial frequencies beyond the diffraction limit with much greater strength than do other alternatives and hence require far fewer photons emitted from the specimen, using far less intense light.
CONCLUSION
High-NA TIRF-SIM and PA NL-SIM fill an unmet need for minimally invasive tools to image live cells in the gap between the 100-nm resolution traditionally associated with SIM and the sub-60-nm regime of protein-specific structural imaging served by single-molecule localization microscopy.
Two approaches for improved live-cell imaging at sub-100-nm resolution.
(
Left
) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (
inset
) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (
Right
) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). (Left and right represent single frames from time-lapse movies over 91 and 30 frames, respectively. Scale bars, 2 μm (left); 3 μm (right).
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be ...coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.
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